EP1470227B1 - Procédé d'introduction sélective d'une double liaison dans un stéroide - Google Patents

Procédé d'introduction sélective d'une double liaison dans un stéroide Download PDF

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EP1470227B1
EP1470227B1 EP03704484A EP03704484A EP1470227B1 EP 1470227 B1 EP1470227 B1 EP 1470227B1 EP 03704484 A EP03704484 A EP 03704484A EP 03704484 A EP03704484 A EP 03704484A EP 1470227 B1 EP1470227 B1 EP 1470227B1
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seq
promoter
bacillus
steroid
terminator
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EP1470227A2 (fr
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Tilman Spellig
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Bayer Intellectual Property GmbH
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Bayer Schering Pharma AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/02—Dehydrogenating; Dehydroxylating

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  • the present invention relates to a method for the selective introduction of a double bond into a steroid skeleton for the overexpression of dehydrogenases, especially ⁇ 1 -dehydrogenases, in particular 3-ketosteroid- ⁇ 1 -dehydrogenases and the plasmids and DNA sequences used for overexpression.
  • dehydrogenases especially ⁇ 1 -dehydrogenases, in particular 3-ketosteroid- ⁇ 1 -dehydrogenases and the plasmids and DNA sequences used for overexpression.
  • 3-Ketosteroid- ⁇ 1 -dehydrogenase is an enzyme that plays an important role in steroid metabolism. With the help of this enzyme, the selective introduction of a double formation at position 1 in the steroid skeleton is made possible.
  • the bacteria mentioned in process steps b) and d) can be added to the Gram- positive genus Bacillus, such as Bacillus spec., Bacillus subtilis, Bacillus sphaericus, Bacillus megaterium, Bacillus licheniformis, Bacillus lentus and to the gram- positive representatives Arthrobacter simplex and Brevibacterium maris or to the gram- negative representatives Escherichia coli and Pseudomonas species .
  • the present invention relates in particular to the Bacillus sphaericus 3-ketosteroid ⁇ 1 -dehydrogenase gene with promoter and terminator elements according to Seq. ID No. 9 or Seq. ID No. 10 and according to Seq. ID No. 11th
  • plasmids which contain at least one of the abovementioned DNA sequences.
  • the ⁇ 1 -dehydrogenase genes are provided with suitable promoters and terminators, which are required for overexpression in bacteria.
  • suitable promoters and terminators are, for example, promoter and terminator of the Bacillus sphaericus 3-keto steroid ⁇ 1 -dehydrogenase gene according to Seq. ID No.
  • constitutive promoters such as p (veg) or promoters of bacteriophage ⁇ 29 and SPO1, inducible promoters such as p (aprE) or p ( sacB ) from Bacillus subtilis, hybrid promoters such as a lacl- controlled SPO1 promoter, terminators of Escherichia coli such as t ( rrnB) or Bacillus subtilis such as t (senS) or t (senN) [ Sua Doi RH (1984) In: Biotechnology and Genetic Engineering Reviews, Vol 2, Russell GE (ed), Intercept, Newcastle Upon Tyne, UK, pp 121 -153; Le Grice SFJ et al.
  • the plasmids are also the subject of the present invention.
  • the plasmids can be used to transform bacteria capable of overexpression of ⁇ 1 -dehydrogenases.
  • the invention also relates to the DNA sequences with 3-ketosteroid ⁇ 1 -dehydrogenase activity whose DNA sequences have a homology of more than 80%, in particular a homology of more than 90% and preferably a homology of more than 95% in Comparison to Seq. ID No. 10 have.
  • the invention also relates to protein sequences with 3-ketosteroid ⁇ 1 -dehydrogenase activity having a homology of at least 90%, in particular a homology of at least 95% in comparison to Seq. ID No. 11 have.
  • the invention also relates to promoters, in particular the Bacillus sphaericus 3-keto steroid ⁇ 1 -dehydrogenase promoter with the DNA sequence Seq. ID. No. 9, as well as homologous promoters that have homology to Seq. ID No. 9 of more than 80%, preferably more than 90% and more preferably more than 95% compared to Seq. ID No. 9 have.
  • the invention further relates to the Bacillus sphaericus 3-keto steroid ⁇ 1 -dehydrogenase oligonucleotides according to the sequences Seq. ID No. 15, Seq. ID No. 16, Seq. ID No.17 and Seq. ID No. 18, and the parS oligonucleotides according to the sequences Seq. ID No. 19 and Seq. ID No. 20th
  • the DNA sequences and proteins of the invention can be used for the selective dehydrogenation of steroids.
  • Dehydrated steroids are, for example, betamethasone, clobetasone, clocortolone, ⁇ 1 -11 ⁇ , 17 ⁇ -dihydroxy-6 ⁇ , 9 ⁇ -difluoro-16 ⁇ -methylprogesterone, deflazacort, dexamethasone, diflocortolone, fluocinolone acetonide, fluocortolone, hydroxy acid and prednisolone and derivatives of said compounds.
  • the bacterial strains mentioned in the application can be obtained from the respective depositories, eg from the DSM ⁇ German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, D-38124 Braunschweig; ATCC ⁇ American Type Culture Collection, Rockville, Maryland, USA; NRRL ⁇ Northern Utilization Research and Development Division, Peoria, Illinois, USA; Etc.
  • Restrictions of plasmid and genomic DNA were carried out in volumes of 15 to 100 ⁇ l, depending on the amount of DNA used [1 to 20 ⁇ g].
  • the enzyme concentration was 1 to 5 units of restriction enzyme per ⁇ g of DNA.
  • the reaction was carried out in a buffer, incubated for one to three hours and then analyzed on an agarose gel [Sambrook et al. (1989) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York].
  • Preparative restriction mixtures were separated by size in the agarose gel. The desired bands were cut out with a scalpel. The DNA fragment to be isolated was recovered using the "Jetsorb Kit” [Genomed] in accordance with the manufacturer's instructions and taken up in TE buffer.
  • oligonucleotide 50 pmol oligonucleotide was incubated at 37 ° C in the manufacturer's recommended buffer in the presence of 0.1 mM ATP and 20 units T4 polynucleotide kinase for 45 min. Enzyme inactivation was at 68 ° C [Sambrook et al. (1989) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York].
  • Ligation was carried out using appropriate amounts of dephosphorylated, linearized vector DNA and fragment DNA in a molar ratio of 1: 5. The reaction was carried out in a volume of 10 ⁇ l with 1 unit T4 DNA ligase in the manufacturer's recommended buffer at 16 ° C overnight in a water bath [Sambrook et al. (1989) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York].
  • Competent E. coli cells were obtained by CaCl 2 treatment and stored at -80 ° C. As a rule, a 10 ⁇ l ligation mixture was incubated with 200 ⁇ l competent cells. The transformation batches were plated on LB agar with the antibiotic supplement required in each case and incubated at 37 ° C. for 16 hours. Competent cell production and transformation were performed according to Sambrook et al. (1989) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • Bacillus subtilis was carried out according to the two-step method described by Cutting SM and Vander Horn PB [In: Molecular Biological Methods for Bacillus (1990), Harwood CR and Cutting SM (eds), John Wiley & Sons, Chichester].
  • Bacillus sphaericus was transformed by electroporation in accordance with a method published by Taylor and Burke (1990) [FEMS Microbiol Lett 66: 125-128].
  • Cells were harvested in MM2G medium [0.3% (w / v) meat extract, 0.8% (w / v) yeast extract, 1% (w / v) peptone, 0.2% (w / v) glucose, 0.7% (w / v) NaCl, 7.36 g / LK 2 HPO 4 , 2.65 g / L KH 2 PO 4 , 5 ml / L 100% glycerol, pH 7] were grown overnight, transferred 1:20 to fresh MM2G medium and incubated at 37 ° for 90 min C and 250 rpm cultivated.
  • the cells were pelleted, washed 3 times with 10% glycerol and then taken up in 750 ⁇ l glycerol.
  • 50 ul of cell suspension were in a Electroporation cuvette mixed with plasmid DNA, incubated on ice and placed in the electroporation device [Biorad Gene Pulser TM ] [2.5 kV, 25 ⁇ F, 600 ⁇ ].
  • Cells were incubated for 90 min at 30 ° C in MM2G medium followed by plating on TBAB agar / 5 ⁇ g neomycin [Tryptose Blood Agar Base (Difco)] and incubated at 30 ° C for 24 h.
  • the preparation of plasmids from Bacillus subtilis and Bacillus sphaericus was carried out on columns from Genomed ["Jetstar Kit Mini"] according to the manufacturer's protocol.
  • the cell pellet taken up in buffer E1 was admixed with 5 mg / ml lysozyme and the cells were incubated for one hour at 37.degree.
  • Plasmid Maxigraphy was carried out with the "Jetstar Kit Maxi" from Genomed. The strains were grown in 200 ml of LB medium in the presence of an antibiotic overnight. The preparation of the plasmids was carried out according to the protocol given by the manufacturer. In order to ensure complete lysis of Bacillus subtilis and Bacillus sphaericus , the cell pellets taken up in buffer E1 were additionally admixed with 5 mg / ml lysozyme and the cells were incubated for one hour at 37.degree.
  • the reaction conditions for the PCR were optimized for each individual case. As a rule, 0.1 to 0.5 ⁇ g of template DNA, 10 mM of dNTPs, 50 pmol of 5 'and 3' primers and 2.5 units of Pwo polymerase [Boehringer Mannheim] were added to the buffer recommended by the manufacturer in 100 ml ⁇ l total volume combined. Depending on the template DNA, up to 10% DMSO was added to the batch. The PCR was performed in a "Biometra Trio Thermoblock". The temperature profile was adapted for each research question. The annealing temperature varied between 50 ° C [low stringent conditions] and 65 ° C. [S. PCR 1: A Practical Approach, McPherson et al. (eds), Oxford University Press (1991)]
  • DNA separated by size in the agarose gel was analyzed by the capillary blot method [Sambrook et al. (1989) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York] was transferred to positively charged nylon membranes and covalently linked to the membrane by UV irradiation. Hybridizations were performed with digoxigenin labeled probes. The labeling of the probes was carried out with the "DIG - High - Prime" or the "PCR DIG Sample Synthesis Kit "from Boehringer Mannheim according to the protocol recommended by the manufacturer. For hybridization, an SDS-phosphate buffer was used [7% SDS (w / v); 0.5 M NaPhosphate pH 7.0].
  • DNA sequence analyzes were performed with the GATC ® 1500 system. The sequence reactions were carried out and with the GATC ® -BioCycle Sequencing Kit according to the manufacturer's recommended protocol on a 4% polyacrylamide-urea gel analysis [GATC ® 1500 system protocol]. Detection was performed with CSPD ® [GATC ® -Biocyde Sequencing Kit Protocol].
  • the culture broth was diluted with 3 times the volume of methanol / 1% acetic acid, sonicated and spun off.
  • the supernatant was chromatographed on an ODS Hypersil column [250 x 4.6 mm] with an acetonitrile-water gradient at a flow rate of 1 ml / min. Sequence of eluents: hydrocortisone, prednisolone, 11 ⁇ -hydroxyandrosta-1,4-diene-3,17-dione, hydrocortisone-17-acetate, hydrocortisone-21-acetate, prednisolone-21-acetate.
  • the culture broth was adjusted to pH 4-6 with acetic acid and then extracted with 4 times the volume of isobutyl methyl ketone.
  • the extract was evaporated, taken up in chloroform and on a Kromasil 100 column [250 x 4 mm] with an isocratic gradient of chloroform: isooctane: 1,4-dioxane: ethanol: water 1000: 100: 50: 10: 2 at a flow rate chromatographed at 1.2 ml / min. Sequence of eluents: fluocortolone A acetate, fluocortolone A, fluocortolone
  • the culture broth was diluted with 3 times the volume of methanol / 1% acetic acid, sonicated and spun off.
  • the supernatant was chromatographed on an ODS Hypersil column [250 x 4.6 mm] with an acetonitrile-water gradient at a flow rate of 1 ml / min. Sequence of eluents: 11 ⁇ , 17 ⁇ -dihydroxy-6 ⁇ , 9 ⁇ -difluoro-16 ⁇ -methyl progesterone, ⁇ 1 -11 ⁇ , 17 ⁇ -dihydroxy-6 ⁇ , 9 ⁇ -difluoro-16 ⁇ -methyl progesterone
  • the culture broth was turraxiert and then extracted with 4 times the volume of methyl isobutyl ketone.
  • the extract was evaporated to dryness and taken up in the same volume of chloroform.
  • the sample was applied to a Kromasil-100 column [250 x 4.6 mm] and chromatographed with diisopropyl ether: dichloroethane: 1,4-dioxane: H 2 O (250: 150: 75: 4) at a flow rate of 2 ml / min.
  • overlapping genomic clones were isolated from a DNA library prepared using the Zero Background TM / Kan Cloning Kit [Invitrogen].
  • the sequence of the Bacillus sphaericus 3-keto steroid- ⁇ 1 -dehydrogenase gene was determined using a GATC ® 1500 Sequencer [GATC].
  • the gene sequence derived protein sequence is related to the sequence of 3-ketosteroid ⁇ 1 -dehydrogenase from Comamonas testosteroni identical to 34%. The similarity is 54%.
  • Arthrobacter simplex 3-ketosteroid ⁇ 1 dehydrogenase has an identity of 34% and a similarity of 54%.
  • 3-keto steroid- ⁇ 1 -dehydrogenase gene from Bacillus -Dehydrogenasegene 1 were used sphaericus promoter and terminator elements of the 3-keto steroid- ⁇ . Both elements were isolated and characterized during cloning of the gene.
  • the promoter contains at position 84 bp or 61 bp above the start codon two hexanucleotides [TTGACT-84 to -79 / TATACT- 61bis-56 ], which correspond with one deviation to the consensus of bacterial promoters [-10 / -35 box].
  • the distance of 17 nucleotides of the two elements corresponds exactly to the bacterial consensus [s.
  • pUB110 was restricted with Eco RI and Pvu III and the resulting 3.6 kb fragment was inserted into the Eco RI and Eco RV cleavage sites of pSP72.
  • Bacillus sphaericus 3-ketosteroid ⁇ 1 -dehydrogenase gene flanked by promoter and termination sequences [position-126 (Sa / l) to position 1861 (Scal)] was ligated into the Xbal and Pvull cleavage sites of the XbaI-Scal fragment ligated as described above [ ⁇ TS # 196, s. Fig. 2].
  • a second expression plasmid carries a modified ⁇ 1 -dehydrogenase gene promoter p ( ⁇ 1 ) mut :
  • ⁇ 1 modified ⁇ 1 -dehydrogenase gene promoter p ( ⁇ 1 ) mut :
  • TTGACT ⁇ TTGACA was exchanged in the -35 [TTGACT ⁇ TTGACA] and in the -10 box [TATACT ⁇ TATAAT] in order to obtain to achieve an exact match with the consensus of bacterial promoters.
  • the promoter was first amplified with the mutagenesis primer 2089 mut [CCA TCG ATG AAT CTG GTC TTC CTA TTA AAA ATT ATA GAA TTA AAC TAA TT TCT GTC AAT TTT TCC -29bis-91 ] and primer 2090 [CAT GAC AAA ATT ATT TGA TTT AAT CAC -258 bis-284 ] and inserted as Pst I Cl a l fragment in the corresponding cleavage sites of pBluescript II KS (+).
  • the mutations were due Sequence analysis verified.
  • p ( ⁇ 1 ) mut was excised as the Xb aI-ClaI fragment and ligated into the appropriate TS # 196 interfaces.
  • the wt promoter was exchanged for p ( ⁇ 1 ) mut [ ⁇ TS # 251].
  • Two additional plasmids additionally carry a plasmid stabilizing signal, parS [Lin DC and Grossman AD (1998) Cell 92 : 675-685]. This was done via two complementary oligonucleotides, 2091 parS [GAT CCT GTT CCA CGT GAA ACA G] and 2092 parS [GAT CCT GTT TCA CGT GGA ACA G], into the Bam HI site of TS # 196 [ ⁇ AD # 82] and TS # 251 [ ⁇ TS # 255] cloned.
  • Bacillus subtilis DSM 402 German strain collection for microorganisms, Braunschweig
  • Bacillus sphaericus ATCC 13805 Bacillus subtilis and Bacillus sphaericus are Gram-positive, non-pathogenic organisms. They are easy to cultivate. In contrast to Bacillus sphaericus Bacillus subtilis is well characterized molecular genetically. There are a number of examples of the heterologous expression and secretion of proteins for the production of recombinant gene products [Wang and Doi (1992) In: Biology of Bacilli: Applications to Industry, Doi et al.
  • Strains AD # 67 TS # 196 ' AD # 94 TS # 251 , AD # 95 TS # 255 , AD # 96 TS # 255 , AO # 116 TS # 251, and AO # 205 TS # 196 are derived from Bacillus sphaericus ATCC 13085 and each contain the indicated expression plasmid. Strains AD # 89 TS # 196 and AD # 90 TS # 196 are derived from Bacillus subtilis DSM 402 and each contain the indicated expression plasmid.
  • Bacillus subtilis DSM 402 and Escherichia coli DH5 ⁇ are not expected to react, Bacillus sphaericus ATCC 13085 produces less than 20% product after 24 hours, while all recombinant strains of the genus Bacillus [AD # 67 TS # 196 , AD # 94 TS # 251 , AD # 95 TS # 255 , AD # 96 TS # 255 , AD # 89 TS # 196, and AD # 90 TS # 196 ] produce more than 80% Pln over the same time period.
  • a ⁇ 1 dehydrogenation using the example of a conversion of EAF / MAF / F to Pln analogously above was carried out at a substrate concentration of 1 g / L in a shake flask [LB medium, 37 ° C, 220 rpm].
  • the substrate was added after 3 hours.
  • samples were taken after 4, 5, 6, 7, 8, 9, 10, 11, 12 and 24 hours, the starting materials and product were extracted and analyzed by HPLC. While the strain ATCC 13805 requires 24 hours to fully convert the substrate into Pln, strain AD # 67 has formed the corresponding amount of Pln already after ⁇ 10 hours [Fig. 3; Reaction scheme see below].
  • strain AD # 67 is not limited to the process of preparing prednisolone from EAF / MAF / F but is generally applicable to the introduction of ⁇ 1 into steroid molecules.
  • Fluorinated steroids are also dehydrogenated much more efficiently at position 1 by recombinant strains than was previously possible with the available biocatalysts. This shows the conversion of FCAA to FC analogously in the top of the shake flask by AD # 116 compared to Bacillus sphaericus ATCC 13805 [Fig , 6; Reaction scheme see below].
  • the ⁇ 1 dehydrogenation capacity of strain AD # 67 in comparison to Bacillus sphaericus ATCC 13805 was tested on the example EAF / MAF / F ⁇ Pln in the 10 L fermenter. The reaction was carried out at a 20 times higher substrate load. The seed material was grown in a first step overnight at 37 ° C. and 220 rpm in LB medium in the presence of 5 ⁇ g / ml neomycin [AD # 67] or without the addition of an antibiotic [ATCC 13805]. Subsequently, the overnight culture was transferred 1: 100 in a 1000 ml intermediate culture and shaken for 9 h at 37 ° C and 220 rpm to an optical density of 2.4.
  • the fermentation was carried out in LB medium without the addition of an antibiotic. in principle however, any other medium can be used in which the organism can grow.
  • the substrate was added continuously after 3 hours over 30 hours.
  • the pH was maintained at pH 8.
  • the fermentation profile shows that Bacillus sphaericus ATCC 13805 can not handle substrate concentrations of this magnitude: The reaction stops at a residual substrate of over 80%.
  • the conversion capacity of strain AD # 67 is considerable: Shortly after completion of the substrate application phase, the reaction has proceeded almost completely [> 98%] [Fig. 8th].
  • the conversion activity of strain AD # 67 is 0.6 g / L per hour.
  • strain ATCC 13805 shows an activity of 0.1 g / L per hour. Disturbing secondary zones such as 11- ⁇ -OH-ADD were at best observed in traces. The crystallizate yield of Pln was more than 80% of theory and corresponds to the value obtained in the conventional process [reaction scheme see below].

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Claims (26)

  1. Procédé pour l'introduction sélective d'une double liaison dans une structure stéroïde par surexpression de déshydrogénases, caractérisé en ce qu'on
    a) isole un gène de déshydrogénase d'une bactérie Bacillus sphaericus, on le clone et on l'amplifie,
    b) isole des éléments de type promoteur et de type terminateur du gène de déshydrogénase ou d'autres éléments de type promoteur et terminateur de la même bactérie ou d'une autre bactérie, on les clone et on les amplifie,
    c) construit des plasmides d'expression, dans lesquels est contenu le gène de déshydrogénase de a), flanqué de la séquence de promoteur et de terminateur du gène de déshydrogénase ou d'autres éléments promoteurs et terminateurs de b),
    d) transforme des bactéries avec le plasmide d'expression préparé au point c) et
    e) cultive les bactéries ainsi préparées et on réalise avec ces cultures la déshydrogénation sélective dans la structure stéroïde,
    i) en utilisant une concentration élevée en substrat avec un temps de fonctionnement inchangé et
    ii) sans formation de zones secondaires gênantes.
  2. Procédé pour l'introduction sélective d'une double liaison dans une structure stéroïde par surexpression de Δ1-déshydrogénases selon la revendication 1, caractérisé en ce qu'on
    a) isole un gène de Δ1-déshydrogénase d'une bactérie Bacillus sphaericus, on le clone et on l'amplifie,
    b) isole des éléments de type promoteur et de type terminateur du gène de Δ1-déshydrogénase ou d'autres éléments de type promoteur et terminateur de la même bactérie ou d'une autre bactérie, on les clone et on les amplifie,
    c) construit des plasmides d'expression, dans lesquels est contenu le gène de Δ1-déshydrogénase de a), flanqué de la séquence de promoteur et de type terminateur du gène de Δ1-déshydrogénase ou d'autres éléments de type promoteur et de type terminateur de b),
    d) transforme des bactéries avec le plasmide d'expression préparé au point c) et
    e) cultive les bactéries ainsi préparées et on réalise avec ces cultures la déshydrogénation sélective dans la structure stéroïde,
    i) en utilisant une concentration élevée en substrat avec un temps de fonctionnement inchangé et
    ii) sans formation de zones secondaires gênantes.
  3. Procédé pour l'introduction sélective d'une double liaison dans une structure stéroïde par surexpression de 3-cétostéroïde-Δ1-déshydrogénases selon la revendication 2, caractérisé en ce qu'on
    a) isole un gène de 3-cétostéroïde-Δ1-déshydrogénase d'une bactérie Bacillus sphaericus, on le clone et on l'amplifie,
    b) isole des éléments de type promoteur et de type terminateur du gène de 3-cétostéroïde-Δ1-déshydrogénase ou d'autres éléments de type promoteur et de type terminateur de la même bactérie ou d'une autre bactérie, on les clone et on les amplifie,
    c) construit des plasmides d'expression, dans lesquels est contenu le gène de 3-cétostéroïde-Δ1-déshydrogénase de a), flanqué de la séquence de promoteur et de terminateur du gène de 3-cétostéroïde-Δ1-déshydrogénase ou d'autres éléments de type promoteur et de type terminateur de b),
    d) transforme des bactéries avec le plasmide d'expression préparé au point c) et
    e) cultive les bactéries ainsi préparées et on réalise avec ces cultures la déshydrogénation sélective en position 1 dans la structure stéroïde,
    i) en utilisant une concentration élevée en substrat avec un temps de fonctionnement inchangé et
    ii) sans formation de zones secondaires gênantes.
  4. Procédé selon l'une quelconque des revendications 1 à 3, caractérisé en ce que les bactéries mentionnées aux points b) et d) appartiennent aux genres gram-positifs Bacillus et Arthrobacter et aux genres gram-négatifs Escherichia coli et Pseudomonas.
  5. Procédé selon la revendication 4, caractérisé en ce que les bactéries appartiennent aux espèces Bacillus spec., Bacillus subtilis, Bacillus sphaericus, Bacillus megaterium, Bacillus licheniformis, Bacillus lentus, Arthrobacter simplex, Brevibacterium maris et aux espèces Pseudomonas.
  6. Gène de 3-cétostéroïde-Δ1-déshydrogénase de Bacillus sphaericus selon la SEQ. ID. No. 10.
  7. 3-cétostéroïde-Δ1-déshydrogénase de Bacillus sphaericus selon la SEQ. ID. No. 11.
  8. Promoteur du gène de 3-cétostéroïde-Δ1-déshydrogénase de Bacillus sphaericus selon la SEQ. ID. No. 9.
  9. Plasmides, contenant au moins une séquence d'ADN selon l'une quelconque des revendications 6 et 8.
  10. Plasmides selon la revendication 9,
    caractérisés en ce qu'ils contiennent des promoteurs et des terminateurs appropriés pour la surexpression de Δ1-déshydrogénases dans les bactéries.
  11. Plasmides selon la revendication 10,
    caractérisés en ce qu'on utilise comme terminateurs le terminateur du gène de 3-cétostéroïde-Δ1-déshydrogénase de Bacillus sphaericus selon la SEQ. ID. No. 10, ou des terminateurs d'Escherichia coli ou de Bacillus subtilis et comme promoteurs le promoteur du gène de 3-cétostéroïde-Δ1-déshydrogénase de Bacillus sphaericus selon la SEQ. ID. No. 9, ou des promoteurs constitutifs
    ou des promoteurs des bactériophages ϕ29 et SPO1, ou des promoteurs pouvant être induits de Bacillus subtilis ou des promoteurs hybrides.
  12. Plasmides selon la revendication 11,
    caractérisés en ce qu'on utilise comme terminateur le terminateur d'Escherichia coli t(rmB), le terminateur de Bacillus subtilis t (senS) ou t(senN) et comme promoteur le promoteur constitutif p(veg), comme promoteur pouvant être induit le promoteur p(aprE) ou p(sacB) de Bacillus subtilis ou comme promoteur hybride un promoteur SPO1 contrôlé par lacl.
  13. Utilisation des plasmides selon les revendications 9 à 12, pour la transformation de bactéries qui sont aptes à la surexpression de Δ1-déshydrogénases.
  14. Séquences d'ADN selon la revendication 6, qui codent pour une protéine avec une activité 3-cétostéroïde-Δ1-déshydrogénase, et dont les séquences d'ADN présentent une homologie supérieure à 80% par rapport à la SEQ. ID. No. 10.
  15. Séquences d'ADN selon la revendication 6, qui codent pour une protéine avec une activité 3-cétostéroïde-Δ1-déshydrogénase, et dont les séquences d'ADN présentent une homologie supérieure à 90% par rapport à la SEQ. ID. No. 10.
  16. Séquences d'ADN selon la revendication 6, qui codent pour une protéine avec une activité 3-cétostéroïde-Δ1-déshydrogénase, et dont les séquences d'ADN présentent une homologie supérieure à 95% par rapport à la SEQ. ID. No. 10.
  17. Séquences de protéine selon la revendication 7, qui présentent une homologie d'au moins 90% par rapport à la SEQ. ID. No. 11 et qui possèdent une activité de 3-cétostéroïde-Δ1-déshydrogénase.
  18. Séquences de protéine selon la revendication 7, qui présentent une homologie d'au moins 95% par rapport à la SEQ. ID. No. 11 et qui possèdent une activité de 3-cétostéroïde-Δ1-déshydrogénase.
  19. Séquence d'ADN de promoteur selon la revendication 8, dont la séquence d'ADN présente une homologie supérieure à 80% par rapport à la SEQ. ID. No. 9.
  20. Séquence d'ADN de promoteur selon la revendication 8, dont la séquence d'ADN présente une homologie supérieure à 90% par rapport à la SEQ. ID. No. 9.
  21. Séquence d'ADN de promoteur selon la revendication 8, dont la séquence d'ADN présente une homologie supérieure à 95% par rapport à la SEQ. ID. No. 9.
  22. Oligonucléotide de 3-cétostéroïde-Δ1-déshydrogénase de Bacillus sphaericus selon les séquences SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 17 et SEQ. ID. No. 18.
  23. Oligonucléotide parS selon les séquences SEQ. ID. No. 19 et SEQ. ID. No. 20.
  24. Utilisation des protéines codées par les séquences d'ADN selon la revendication 6 et des protéines codées par les séquences d'ADN selon les revendications 14 à 16, pour la déshydratation sélective de stéroïdes.
  25. Utilisation des protéines selon les revendications 7, 17 et 18 pour la déshydrogénation sélective de stéroïdes.
  26. Utilisation selon les revendications 24 et 25, caractérisée en ce que le stéroïde déshydrogéné est le Betamethason, le Clobetason, le Clocortoton, la Δ1-11β,17α-dihydroxy-6α,9α-difluoro-16α-méthylprogestérone, la 11β,21-dihydroxy-2'-méthyl-5'βH-pregn-4-éno[17,16-d]oxazol-3,20-dione, le Deflazacort, l'alcool de Deflazacort, le Dexamethason, le Diflocortolon, le Fluocinolonacetonid, le Fluocortolon, l'hydroxyacide et le Prednisolon.
EP03704484A 2002-02-01 2003-01-28 Procédé d'introduction sélective d'une double liaison dans un stéroide Expired - Lifetime EP1470227B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10204798A DE10204798A1 (de) 2002-02-01 2002-02-01 Verfahren zur Überexpression von Dehydrogenasen
DE10204798 2002-02-01
PCT/EP2003/000855 WO2003064653A2 (fr) 2002-02-01 2003-01-28 Procede de sur-expression de dehydrogenases

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EP1470227A2 EP1470227A2 (fr) 2004-10-27
EP1470227B1 true EP1470227B1 (fr) 2007-04-11

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CN100374570C (zh) * 2004-07-07 2008-03-12 新疆农业科学院微生物应用研究所 一种将穿梭质粒导入黄色短杆菌的电转化方法
US20110191875A1 (en) * 2007-11-16 2011-08-04 Verenium Corporation Compositions and methods for making androstenediones
CN106893753A (zh) * 2015-12-21 2017-06-27 天津金耀集团有限公司 一种通过生物发酵一步制备泼尼松龙的方法
CN115927428A (zh) * 2022-08-25 2023-04-07 天津科技大学 一种应用于简单节杆菌的基因快速编辑方法、遗传操作系统、菌株和应用

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FR2657882B1 (fr) * 1990-02-07 1994-08-12 Centre Nat Rech Scient Fragments d'adn comprenant le gene de la 1-deshydrogenase, micro-organismes transformes par lesdits fragments et applications desdits fragments et micro-organismes.

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WO2003064653A3 (fr) 2003-12-24
JP2006502692A (ja) 2006-01-26
WO2003064653A2 (fr) 2003-08-07
AU2003206792A1 (en) 2003-09-02
EP1470227A2 (fr) 2004-10-27
ES2285095T3 (es) 2007-11-16
DE10204798A1 (de) 2003-08-14
PT1470227E (pt) 2007-07-23
JP4460300B2 (ja) 2010-05-12
DE50307011D1 (de) 2007-05-24
ATE359368T1 (de) 2007-05-15

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